Support carrying an immobilized selective binding substance

ABSTRACT

A support carrying an immobilized selective binding substance, that the support surface has a polymer containing the structural unit represented by the following General Formula (1) in an amount of 10% or more with respect all monomer units, and a selective binding substance is immobilized on the support surface by binding to the carboxyl group formed thereon via a covalent bond: 
     
       
         
         
             
             
         
       
     
     (in General Formula (1), R 1 , R 2 , and R 3  each represent an alkyl or aryl group or a hydrogen atom.)

RELATED APPLICATION

This application is a divisional of U.S. patent application Ser. No.10/557,070, filed Nov. 15, 2005′ and accorded a filing date under§371(c) of Dec. 9, 2005, which is a §371 of International ApplicationNo. PCT/JP2004/07060, with an international filing date of May 18, 2004(WO 2004/102194 A1, published Nov. 25, 2004), which is based on JapanesePatent Application No. 2003-140016, filed May 19, 2003 and JapanesePatent Application No. 2003-417661, filed Dec. 16, 2003, the contents ofall of which are herein incorporated by reference.

TECHNICAL FIELD

This disclosure relates to a support carrying an immobilized substancethat binds to an analyte substance selectively (“selective bindingsubstance” in the present specification).

BACKGROUND

Along with advance in research on genetic information analysis ofvarious living organisms, more information about a number of genesincluding human genes and their base sequences as well as the proteinscoded by the gene sequences and the sugar chains produced there proteinssecondarily is becoming available rapidly. The functions of themacromolecules such as gene, protein, and sugar chain with distinctsequence can be studied by various methods. For example as for nucleicacids, mainly, the relationship between various genes and theirbiological functions can be studied for example by using thecomplementarity of a nucleic acid and another nucleic acid such asNorthern hybridization or Southern hybridization. As for proteins, it ispossible to study the function and expression of proteins such bymethods of using protein/protein interaction such as Westernhybridization.

In recent years, a new assay method or methodology called DNA microarraymethod (DNA chip method) was developed and attracting attention recentlyas a method of analyzing expression of multiple genes at the same time.All these methods are the same in principle as conventional methods,because they are methods of detecting and quantifying nucleic acidsbased on hybridization reaction between nucleic acids, and applicable todetection and quantification of proteins and sugar chains based on theinteraction between protein/protein, sugar chain/sugar chain, or sugarchain/protein. These methods are characteristic in that a piece of flatglass substrate called microarray or chip carrying multiple DNAfragments, proteins, or sugar chains that are immobilized densely isused. Typical examples of the use of the microarray method include amethod of hybridizing a gene expressed in analyte cell with a samplelabeled, for example, a fluorochrome on a flat substrate, allowingmutually complementary nucleic acids (DNA or RNA) to bind to each other,and detecting the binding sites rapidly in a high-resolution analyzer;and a method of detecting the response such as the change in electriccurrent due to an electrochemical reaction. In this manner, it ispossible to estimate the amounts of the genes present in sample.

For example, Japanese Patent Application National Publication(Laid-Open) No. 10-503841 (Claims) discloses a method of coatingpoly-L-lysine, aminosilane, or the like on a flat substrate such asslide glass and immobilizing nucleic acids by using a spotting devicecalled spotter, as the method for immobilizing a nucleic acid onsubstrate.

Alternatively, for example, Japanese Patent Application Laid-Open (JP-A)No. 2001-108683 discloses a method of using oligo-DNAs (oligo-DNA is aDNA having a base number of 10 to 100) as nucleic acid probes used onDNA chip (nucleic acids immobilized on substrate) instead of theconventional method of using cDNAs and the fragments thereof having baselengths of hundreds of thousands, for reduction of error duringdetection and convenience of synthesis in synthesizer. In the method,the oligo-DNAs are bound to the glass plate covalently.

In addition to glass, there are some proposals on resin substrates, asthe material for used as the substrate for DNA chip. For example, JP-ANo. 2001-337089 (Paragraph 17) describes a polymethyl methacrylatepolymer. However, JP-A No. 2001-337089 discloses no specific method ofimmobilizing DNA. In addition, JP-A No. 2003-130874 (Paragraph 12) alsohas a similar description but discloses no specific method ofimmobilizing DNA. JP-A No. 2002-71693 (Paragraph 7) discloses a methodof modifying a nitrile group-containing fiber such as acryl fiber into acarboxyl group-containing fiber by alkali treatment and immobilizingDNAs or the like by bonding the carboxyl groups. However, the alkalifiber, which contains polyacrylonitrile as the main component, has aproblem that it has a high autofluorescence as it is and is not suitableas a substrate. Further, JP-A No. 2002-71693 (Paragraph 7) discloses amethod of modifying polymethacrylate by copolymerization with acrylicacid or methacrylic acid into a carboxyl group-containing fiber andallowing the carboxyl groups to bind to DNAs, but the method wasdisadvantageous in that the substrate (support) had a smaller amount ofsurface carboxyl groups leading to decrease in the amount of immobilizedDNA and consequently insufficient intensity of the signal afterhybridization.

SUMMARY

Problems to overcome of this disclosure are resolution of the following:First, immobilization of an oligo-DNA on a flat glass substrate leads tothe following problems: 1) A problem that sample DNA is often adsorbednonspecifically in the area other than the spots where probe DNA isimmobilized during hybridization because the glass is hydrophilic andthe nonspecifically adsorbed sample is also detected during fluorescentdetection with a device called scanner, resulting in increase in noise.2) A problem that the hybridization efficiency with sample DNA is low,the signal intensity is low, and consequently the S/N ratio isinsufficient, probably because the spatial degree of freedom of thecovalently bound oligo-DNA is restricted due to the rigidity of glass.

One of our objectives is to provide a support carrying an immobilizedDNA firmly bound to a resin substrate in a state higher in hybridizationefficiency. Another object thereof is to provide a support carrying animmobilized selective binding substance that is resistant to thedeterioration in the S/N ratio and is higher in detection sensitivity.

Namely, we provide a support carrying an immobilized selective bindingsubstance, which comprises the surface of the support is comprising alow-autofluorescence resin, the surface of the polymer is treated withan alkali or acid, forming carboxyl groups, and additionally a selectivebinding substance is immobilized thereon.

We also provide a support carrying an immobilized selective bindingsubstance which comprises the support that carries the selective bindingsubstance immobilized on the surface thereof, the support surface has apolymer containing the structural unit represented by the followingGeneral Formula (1), the surface of the polymer is treated with analkali or acid and a selective binding substance is immobilized thereon:

(in General Formula (I), R¹, R², and R³ each represent an alkyl or arylgroup or a hydrogen atom.)

We provide a support carrying an immobilized selective binding substancesmaller in adsorption of nonspecific sample, higher in hybridizationefficiency, and consequently superior in S/N ratio.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart illustrating the reaction scheme for immobilizing aselective binding substance on a PMMA surface.

FIG. 2 is a schematic view illustrating a support.

FIG. 3 is a schematic cross-sectional view of the support.

FIG. 4 is a chart illustrating a jig for holding a microarray.

FIG. 5 is a cross-sectional view of the concavo-convex part.

FIG. 6 is a schematic diagram illustrating a support having a supportlayer and a layer carrying an immobilized selective binding substance.

FIG. 7 is chart illustrating the reaction scheme for immobilization of aselective binding substance on a glass surface.

DETAILED DESCRIPTION

Hereinafter, the support carrying an immobilizing a selective bindingsubstance will be described.

The support carrying an immobilized selective binding substancecomprises the surface of the support is comprising alow-autofluorescence resin and the surface of the resin is treated withan alkali or acid, forming carboxyl groups. The low-autofluorescenceresin is a resin having a fluorescence intensity of 1,000 or less, asdetermined by measuring a clean flat plate thereof having a thickness of1 mm by using GenePix 4000B manufactured by Axon Instruments under theconditions of a excitation wavelength of 532 nm, a set photomultipliergain of 700, and a laser power of 33%. Resins unsatisfying therequirement above are undesirable because of the deterioration in S/Nratio during detection. Examples of such resins include the polymersrepresented by the following General Formula (1).

The support carrying an immobilized selective binding substance has asupport surface of a solid having a polymer containing the structuralunit represented by the following General Formula (1) for immobilizationof a selective binding substance.

in General Formula (1), R¹, R² and R³ each represent an alkyl or arylgroup or a hydrogen atom.)

The polymer may be a homopolymer or a copolymer. At least one type ofmonomer is used as the raw material for the polymer, and the monomer ispresent as a double bond for polymerization or a functional group forpolycondensation, ketone or carboxylic acid or the derivative thereof.The polymer more preferably has the structure represented by GeneralFormula (1).

When the polymer is a copolymer, the polymer preferably contains thestructural unit represented by General Formula (1) in an amount of 10%or more with respect to the total monomer units. When the content of thestructural unit represented by General Formula (1) is 10% or more, it ispossible to form more carboxyl groups on the surface and immobilize moreprobe nucleic acids in the steps described below, leading to improvementin the S/N ratio.

The polymer is a compound having a number-averaged polymerization degreeof 50 or more. The number-averaged polymerization degree of the polymeris preferable in the range of 100 to 10,000, and particularly preferably200 or more and 5,000 or less. The number-averaged polymerization degreecan be determined easily by measuring the molecular weight of a polymeraccording to a common method by GPC (gel permeation chromatography).

In General Formula (1), R¹ and R² each represent an alkyl or aryl groupor a hydrogen atom, and may be the same as or different from each other.The alkyl group may be a straight-chain or branch group, and preferablyhas 1 to 20 carbon atoms. The aryl group preferably has 6 to 18 carbonatoms, more preferably 6 to 12 carbon atoms. The functional group X isselected arbitrarily from O, NR³, and CH₂. R³ is a functional groupdefined similarly to R¹ and R².

Favorable examples of the polymers containing the functional groupsdescribed above include polyalkyl methacrylates (PAMA) such aspolymethyl methacrylate (PMMA), polyethyl methacrylate (PEMA) andpolypropyl methacrylate, and the like. Among them, polymethylmethacrylate is preferable, from the points of processability duringinjection molding or embossing and relatively higher glass transitiontemperature. Alternatively, polyvinyl acetate, polycyclohexylmethacrylate or polyphenyl methacrylate, or the like may also be used.Yet alternatively, a copolymer in combination of the polymer componentsabove or a copolymer in combination of the polymer components and one ormore other polymer components may also be used. The other polymer is,for example, polystyrene.

When a copolymer is used, the ratio of the carbonyl group-containingmonomer, for example, alkyl methacrylate, is preferably in the range of10 mole % or more, because it is possible to increase the amount of thecarboxylic acid groups formed on the surface, raise the capacity forimmobilizing probe nucleic acids, and consequently raise the S/N ratio.The ratio of the monomer in the polymer structural units is morepreferably 50 mole % or more.

Further, it is necessary to conduct a pretreatment of the support withan alkali or acid for immobilizing a selective binding substancecontaining the polymer having at least one structural unit representedby General Formula (1).

In this manner, it becomes possible to form carboxyl groups on thesupport surface. For forming carboxyl groups on the support surface, thealkali or acid treatment may be used alone or in combination withanother method such as sonication while warm or exposure to oxygenplasma, argon plasma or radiation ray. Among these methods, preferablefrom the points of reduction in the damage of the support and easierprocessability is the method of forming carboxyl groups on surface byimmersing the support in a heated alkali or acid. More specifically, thesupport may be immersed in an aqueous sodium hydroxide or sulfuric acidsolution (preferable concentration: 1 to 20N) preferably at atemperature of 30° C. to 80° C. for 1 to 100 hours.

Formation of the carboxyl groups on the support surface by theabove-mentioned method can be confirmed by XPS (X-ray photoelectronspectroscopy). Specifically, the carboxyl groups are labeled withfluorine by using a fluorine-containing labeling reagent (e.g.,trifluoroethanol). It is possible then to estimate the amount of thefunctional groups, from the intensity of Cls and Fls peak areas of thelabeled subsequent sample, taking the reaction rate into consideration.For further improvement in accuracy, formation of the carboxyl groups onthe support surface may be confirmed by determining the fluorinedistribution on the surface of a trifluoroethanol-labeled sample byTOF-SIMS (time-of-flight secondary ion mass spectrometry).

Thus the carboxyl groups are formed on the support surface, it would bepossible to immobilize a selective binding substance on the support, byavidin-biotin interaction by modifying the support with biotin or avidinand additionally modifying the selective binding substance with avidinor biotin, or alternatively, to immobilize a selective bindingsubstance, by allowing the support to react with a linker such asethylenediamine and additionally the linker to react with the selectivebinding substance. However, these methods, which demand two-stepreactions, often result in decrease in the amount of the selectivebinding substance immobilized on the support, because of their reactionyields. Therefore, it is preferable to immobilize a selective bindingsubstance by allowing the carboxyl groups on support to react directlywith the functional group of the selective binding substance. In otherwords, it is preferable to immobilize a selective binding substance bycovalent bonds between the amino or hydroxyl group of the selectivebinding substance and the carboxyl group on the support surface.Generally, various condensation agents such as dicyclohexylcarbodiimideand N-ethyl-5-phenylisoxazolium-3′-sulfonate were used for facilitatingthe bond-forming reaction. Among them, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), which is less toxic and easily removed from thereaction system, is one of the condensation agents most effective forthe condensation reaction of a selective binding substance with thecarboxyl groups on the support surface. Another favorable condensationagent is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholiniumchloride (DMT-MM). The condensation agent, for example EDC, may be usedas it is mixed into a solution of the selective binding substance, oralternatively, a support carrying carboxyl groups previously formed onthe surface is immersed in a solution of EDC and thus the surfacecarboxyl groups are activated.

When the carboxyl groups on support surface are reacted with the aminogroup of a selective binding substance by using such a condensationagent, the selective binding substance is immobilized on the supportsurface by amide bond, while when the carboxyl groups on the supportsurface are reacted with the hydroxyl group of a selective bindingsubstance, the selective binding substance is immobilized on the supportsurface by ester bond. The temperature of the reaction between a samplecontaining the selective binding substance and a support is preferably5° C. to 95° C. and more preferably 15° C. to 65° C. The processingperiod is generally 5 minutes to 24 hours, and preferably 1 hour ormore. FIG. 1 is a chart showing the scheme for immobilizing a selectivebinding substance. (In FIG. 1, 1 represents a PMMA substrate; and 2represents a selective binding substance (DNA).)

By immobilizing a selective binding substance on a polymer surface bythe method described above, it is possible to immobilize the selectivebinding substance densely and rigidly via covalent bonds suppressingnonspecific adsorption of sample (typically, DNA) because of thenegatively charged carboxyl groups present on the surface except in thespotted regions, and to obtain a support higher in hybridizationefficiency with sample, presumably because the immobilized selectivebinding substance has a spatial degree of freedom greater than thatimmobilized on a glass surface. The higher spatial degree of freedomdescribed above provides an advantage that it is possible to obtain anextremely improved hybridization efficiency with sample in particularwhen the immobilized selective binding substance used is a so-calledoligo-DNA having a length of 10 to 100 bases.

By using a polymer having the structural unit represented by GeneralFormula (1) for the support, it is possible to produce a support havinga fine shape more easily in a greater amount, for example, by injectionmolding or hot embossing than from glass, ceramic, or metal. The shapeof the support on which the selective binding substance is immobilizedwill be described below. The support carrying an immobilized selectivebinding substance has a concavo-convex part preferably, and theselective binding substance is preferably immobilized on the face of theprojections. Such a structure eliminates detection of nonspecificallyadsorbed samples as will be described below during analysis, andprovides a support carrying an immobilized selective binding substancethat has smaller noise and consequently is favorable in the S/N ratio.As for the height of multiple projections in the concavo-convex part,the faces of projections are preferably almost the same in height. Theheight almost the same described above means a height that does notcause any significant difference in fluorescence intensity level when afluorescent labeled analyte is allowed to react with a selective bindingsubstance immobilized on the surfaces of projections slightly differentin height and the bound analytes on respective surfaces are scanned witha scanner. Concretely, the height almost the same mean a difference inheight of 100 μm or less. In addition, the support preferably has a flatarea. A typical example is shown in FIGS. 2 and 3. 11 represents theflat area, and a selective binding substance (e.g., nucleic acid) isimmobilized on the surface of the projections represented by 12 in theconcavo-convex part. The top face of the projections in theconcavo-convex part is preferably substantially flat. The term“substantially flat” means that the projection top face does not have anirregularity of 50 μm or more in height. Further, the height of theprojections in the concavo-convex part is almost the same as that of theflat area. The phrase “the height of the flat area is almost the same asthat of the irregularly surfaced area” means that there is nosignificant problem of the decline in signal level when the support isscanned with a scanner. Concretely, the height almost the same means thedifference between the heights of the projection top face in theconcavo-convex part and that of the flat area is less than 100 μm.

Generally on microarrays, a fluorescent-labeled sample and a selectivebinding substance immobilized on a support are allowed to react eachother, and the resulting fluorescence is read out with a device calledscanner. The scanner deflects an excitation laser beam with an objectlens and focuses the laser beam. The focused beam is irradiated onto themicroarray surface, and the focal point of the laser beam is directed tothe microarray surface. The fluorescence generated on the microarray isthen read out by scanning the object lens or the microarray per se underthe same condition.

When a support carrying a selective binding substance immobilized on theprojection top face is scanned by using a scanner, an effect of thefluorescence (noise) of a sample DNA nonspecifically attracted on theconcavo area in the concavo-convex part being hard to detect isdemonstrated. It is because the laser beam is focused on the projectiontop face and defocused in the concavo area. In other words, among themultiple projections on which the selective binding substance isimmobilized, the difference in height between the faces of highest andlowest projections is preferably 50 μm or less. It is becausefluctuation in the heights of the projection top faces greater than thedifference above may prohibit accurate determination of the fluorescenceintensity because of the focal depth of the scanner.

The difference in height between the faces of highest and lowestprojections among the multiple projections on which the selectivebinding substance may be preferably 50 μm or less, more preferably 30 μmor less, and still more preferably, the height of the projections is thesame. The same height in this patent application includes the errors dueto the fluctuations that may occur during production or the like.

The multiple projection area on which the selective binding substance isimmobilized means an area where a selective binding substance essentialfor data collection (e.g., nucleic acid) is immobilized and does notinclude an area where only a dummy selective binding substance isimmobilized.

Generally, the method of adjusting focal point of scanner is as follows:Namely, when focusing an excitation beam on the microarray surface, thescanner adjust the focal point of the laser beam by focusing theexcitation beam at the corner of the microarray or by fixing themicroarray to a jig as shown in FIG. 4. The scanner scans the entiremicroarray under the same condition. (In FIG. 4, 13 represents amicroarray; 14, an object lens; 15, an excitation light; and 16, aspring for fixing a microarray to a jig). Thus, the support preferablyhas a concavo-convex part as well as a flat area. A typical examplethereof is shown in FIGS. 2 and 3. 11 represents a flat area, and aselective binding substance (e.g., nucleic acid) is immobilized on thesurface of the projections in the concavo-convex part represented by 12.Further, the difference in height between the face of the projections inthe concavo-convex part and of that in the flat area is preferably 50 μmor less. In this manner, when a support carrying an immobilizedselective binding substance is scanned, it becomes possible to adjustthe focal point of the excitation beam once on the face of the flat areaor to fix the flat area to the jig, making it easier to position thefocal point of scanner. Because the excitation beam is thus focused inthe flat area, the top face of the projections on which a selectivebinding substance is immobilized is preferably flat and the differencein height between the face of the projections and the face of the flatarea is preferably 50 μm or less.

A difference in height between the face of the projections and the faceof the flat area at 50 μm or more may cause the following problems.Namely, because the focal point of excitation beam is adjusted at thetop face in the flat area, when the height of the projection top facediffers, the focal point of excitation beam may become blurred on theprojection top face, in the worst case resulting in completely nodetection of the fluorescence generated by the reaction between theselective binding substance and the sample. A similar phenomenon mayoccur when there is no flat area that is as high as the face of theprojections.

Alternatively, when the top face of projections is not flat, the focalsize of the excitation beam on the projection top face may vary,consequently leading to fluctuation in the fluorescent intensitydetected even on a single projection top face, which makes thesubsequent analysis more difficult. In the case of present application,such problems are prevented, favorable signal (fluorescence) can beobtained.

The difference in height between the face of the projections and theface of the flat area may be preferably 50 μm or less, more preferably30 μm or less, still more preferably the same as that in the flat area.The same height in this patent application includes the errors due tothe fluctuations that may occur during production or the like.

A selective binding substance is not spotted on a planar support, but aselective binding substance is immobilized only on the face of theprojections in the concavo-convex part. Thus, even when an analytesample is adsorbed nonspecifically onto the area other than theprojection top face, no fluorescence from the undesirable analyte sampleadsorbed nonspecifically is detected, because the focal point ofexcitation beam is blurred in the area other than the projection topface, leading to reduction in noise and consequently improvement of itsS/N ratio.

Use of an injection molding method is desirable from productivity forproduction of the support in such a shape. A mold would be needed forproduction of the support in the above-mentioned shape by the injectionmolding method, and use of a LIGA (Lithographie GalvanoformungAbformung) process is preferable for production of the mold, because itgives a mold from which the molded support is easily separated.

In addition, the areas of the respective top faces of projections arepreferably almost the same. In this manner, it becomes possible to makeuniform the areas on which different selective binding substances areimmobilized, which is advantageous for the subsequent analysis. Thephrase “the areas of the respective top faces of projections arepreferably almost the same” means the value of the largest top face areadivided by the smallest top face area of all the projections is 1.2 orless.

The area of the projection top face is not particularly limited, but ispreferably 4 mm² or less and 10 μm² or more, from the points ofreduction in the amount of the selective binding substance used andeasier handling.

The height of the projections in the concavo-convex part is preferably0.01 mm or more and 1 mm or less. A projection height of lower than thevalue above may result in detection of the nonspecifically adsorbedanalyte sample in the area other than the spots and consequently indeterioration in S/N ratio. Alternatively, a projection height of 1 mmor more may cause problems such as the vulnerability to breakage due tofracture of the projections.

It is also preferable that a conductive material is formed at least onthe side face of the projections. In this manner, it becomes possible toaccelerate hybridization, for example, of nucleic acids, by forming acounter electrode and applying a voltage between the counter electrodeand the conductive material. Preferable area of the projections wherethe conductive material is coated is the entire area of concave parts orthe entire side face of the projections. FIG. 5 shows an example (inFIG. 5, 21 represents the face of a projection; 22, a conductivity film;and 23 an insulation film). If electric current flows, the voltageapplied is preferably in the range of 0.01 V or more and 2 V or less,and particularly preferably in the range of 0.1 V or more and 1.5 V orless. Application of a voltage larger than that above may result inelectrolysis of water and thus generation of adverse effects on theselective binding substance on surface. The material for the conductivematerial is not particularly limited, and example thereof includecarbon, manganese, aluminum, silicon, titanium, vanadium, chromium,manganese, iron, cobalt, nickel, copper, tin, zirconium, niobium,molybdenum, palladium, silver, hafnium, tantalum, tungsten, platinum,gold, stainless steel, or the mixture thereof, and conductive polymers.Among them, platinum, gold, and titanium are used particularlypreferably. Methods of producing the film of these conductive materialsinclude vapor deposition, sputtering, CVD, metal plating, and the like.

When a conductive material is coated on the projection area as describedabove, an insulating material layer is preferably formed additionally onthe area of the projections other than the top face. Presence of theinsulating material layer allows attraction of the analyte onto theprojection top face when an electric current is applied. Examples of theinsulating materials include metal oxides (e.g., Al—O, SiO₂, TiO₂, VO,SnO, Cr—O, Zn—O, GeO₂, Ta₂O₅, ZrO₂, Nb—O, Y₂O₃, etc.), nitrides (Al—N,Si₃N₄, TiN, Ta—N, Ge—N, Zr—N, NbN, etc.), sulfides (e.g., ZnS, PbS, SnS;and CuS), and insulating polymers.

The support carrying an immobilized selective binding substance thusobtained may be treated additionally after immobilization of theselective binding substance. It is possible, for example, to modify theimmobilized selective binding substance by treatment such as heattreatment, alkali treatment, or surfactant treatment.

Generally, a fluorescent labeled sample and a selective bindingsubstance immobilized on a support are allowed to react each other inhybridization reaction on the support carrying an immobilized selectivebinding substance, and the fluorescence form the resulting support isread out with a device called scanner. The scanner deflects anexcitation laser beam with an object lens and focuses the laser beam.However, when there is autofluorescence of the surface of the support,the fluorescence may cause noise and lead to deterioration in detectionaccuracy. For prevention thereof, blackening the surface by adding ablack substance that does not emit light by laser irradiation to thesurface to the polymer having the structural unit represented by GeneralFormula (1) is preferable, as it reduces the autofluorescence from thesupport per se. By using such a support, it is possible to provide asupport carrying an immobilized selective binding substance that haslower noise and consequently a favorable S/N ratio during detection,because the autofluorescence from the support can be reduced.

The blackened support means a support of which the blackened area has auniformly low spectroscopic reflectance not in a particular spectralpattern (e.g., without any particular peaks) and a uniformly lowspectroscopic transmissibility not in a particular spectral pattern inthe visible light range (wavelength: 400 to 800 nm).

As for the spectroscopic reflectance and transmissibility, thespectroscopic reflectance is preferably in the range of 7% or less inthe visible light range (wavelength: 400 to 800 nm) and thespectroscopic transmissibility is preferably 2% or less in the samewavelength range. The spectroscopic reflectance is a spectroscopicreflectance including the regular reflected light from the support, asdetermined in an optical illuminator-detector system compatible with thecondition C of JIS Z8722.

The support may be blackened by adding a black substance to the support,and favorable examples of the black substances include carbon black,graphite, titanium black, aniline black, oxides of metals such as Ru,Mn, Ni, Cr, Fe, Co and Cu, carbides of metals such as Si, Ti, Ta, Zr andCr, and the like.

These black substances may be contained alone or in combination of twoor more; among the black substances, carbon black, graphite, titaniumblack are preferably contained; and carbon black is used particularlypreferably, because it is easily dispersed uniformly in polymer.

As for the shape of support, it is preferable to form a layer carryingan immobilized selective binding substance of a polymer having at leastone structural unit represented by General Formula (1) on a supportlayer of a material resistant to heat deformation such as glass ormetal, for prevention of the alteration in the shape of support due toheat or external force. Alternatively, a resin resistant to relativelyhigh temperature such as polycarbonate, polyimide, or polyamide may beused as the support layer. FIG. 6 is a chart illustrating such astructure. (2 represents a selective binding substance (DNA), 3represents a support layer (glass), and 4 represents a layer carrying animmobilized selective binding substance (PMMA)). Glass or a metal suchas iron, chromium, nickel, titanium, or stainless steel is preferable asthe support layer. In addition, the surface of the support layer ispreferably finished in a plasma treatment with argon, oxygen, ornitrogen gas or treated with a silane-coupling agent, for improvement inadhesion between the support layer and the layer carrying an immobilizedselective binding substance. Examples of the silane-coupling agentsinclude 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane,3-aminopropyldiethoxymethylsilane,3-(2-aminoethylaminopropyl)trimethoxysilane, 3-(2-aminoethylaminopropyl)dimethoxymethylsilane, 3-mercaptopropyltrimethoxysilane,dimethoxy-3-mercaptopropylmethylsilane, and the like. A layer carryingan immobilized selective binding substance is formed on the supportlayer by any one of known means, for example, by spin coating with ordipping in a solution of a polymer dissolved in an organic solvent. Moreconveniently, the layer carrying an immobilized selective bindingsubstance may be adhered to the support with an adhesive.

The “selective binding substance” means a substance that can selectivelybind to an analyte substance directly or indirectly, and typicalExamples thereof include nucleic acids, proteins, saccharides, and otherantigenic compounds. The nucleic acid may be DNA, RNA, or PNA. Singlestrand nucleic acids having a particular base sequence selectivelyhybridizes with and binds to a single strand nucleic acid having thebase sequence complementary to the base sequence or the part thereof,and thus are included in the “selective binding substances.” Examples ofthe proteins include antibodies, antigen-binding antibody fragments suchas Fab fragments and F (ab') 2 fragments, and various antigens.Antibodies and their antigen-binding fragments that selectively bind torespective complementary antigens and antigens that selectively bind torespective complementary antibodies are also included in “selectivebinding substances.” Polysaccharides are preferably as the saccharides,and examples thereof include various antigens. Alternatively, anantigenic substance other than protein or saccharide may be immobilized.The selective binding substance for use may be a commercially availableproduct or a substance prepared from living cell or the like.Particularly preferable as the “selective binding substances” is anucleic acid. Among nucleic acids, so-called oligonucleic acids, nucleicacids having a length of 10 to 100 bases, are preferable, because it ispossible easily to prepare in a synthesizer, attach the amino group tothe terminal of the nucleic acid, and thus immobilize it onto thesupport surface. Further, the length of the oligonucleic acid ispreferably 20 to 100 bases, from the viewpoint that the hybridizationefficiency is lower with an oligonucleic acid having less than 20 bases,and particularly preferably in the range of 40 to 100 bases, forensuring the stability of hybridization efficiency.

Examples of the analyte substances to be processed in the measurementmethod by using the support include, but are not limited to, nucleicacids to be evaluated, such as genes of pathogenic bacteria and virusesand causative genes of genetic diseases, or the partial region thereof;various antigenic biological components; antibodies to pathogenicbacteria and viruses; and the like. Examples of the samples containingthe analyte substances above include, but are not limited to, bodyfluids such as blood, serum, blood plasma, urine, feces, spinal fluid,saliva, and various tissue fluids and various foods and drinks ordiluents thereof, and the like. The analyte nucleic acid may be preparedby labeling a nucleic acid extracted from blood or cell according to acommon method or by amplification by a nucleic acid amplification methodsuch as PCR by using the nucleic acid as a template. In the latter case,it is possible to increase the measurement sensitivity drastically. Whenan amplified nucleic acid product is used as the analyte substance, itis possible to label the amplified nucleic acid by performingamplification in the presence of a nucleotide-3-phosphate labeled with afluorescent material or the like. When the analyte substance is anantigen or antibody, the analyte substance, antigen or antibody, may bedirectly labeled by a common method, or alternatively, the analytesubstance, antigen or antibody, may first bound to a selective bindingsubstance; after washing of the support, the antigen or antibody isallowed react with a labeled antibody or antigen that reacts in theantigen-antibody reaction; and then, the labels bound to the support isanalyzed.

The step for the interaction between the immobilized substance and ananalyte substance may be the same as that traditionally practiced. Thereaction temperature and period may be selected arbitrarily, forexample, according to the chain length of the nucleic acid to behybridized and the kinds of the antigen and/or the antibody involved inthe immune reaction, but the reaction is generally carried out atapproximately 50° C. to 70° C. approximately for 1 minute to more thanten hours in the case of nucleic acid hybridization, and generally, atroom temperature to approximately 40° C. for approximately 1 minute toseveral hours in the case of immune reaction.

EXAMPLES

We describe in more detail with reference to the following Examples, butit should be understood that this disclosure is not restricted by thefollowing Examples.

Referential Example 1

A transparent polymethyl methacrylate (PMMA) plate (Comoglass extrudedplate, manufactured by Kuraray Co., Ltd.; thickness: 1 mm, averagemolecular weight: 150,000, i.e., number-averaged polymerization degree:1,500) was washed thoroughly with ethanol and purified water andimmersed in an aqueous 10N sodium hydroxide solution at 70° C. for 12hours. Then, the plate was washed with purified water, an aqueous 0.1NHCl solution, and purified water in that order. By using thealkali-treated plate and a non-alkali-treated plate as samples, thecarboxyl groups on the sample surface were labeled with afluorine-containing labeling reagent (trifluoroethanol) in gas phase.Then, the samples were analyzed by XPS under the conditions of an X raydiameter of 1 mm and a photoelectron escape angle 90° by usingmonochromatic Al Kα1 and 2 rays (1486.6 eV), and the carboxyl groupsthereon were determined from the Cls and Fls peak area intensities,taking into consideration the reaction rate. The results revealed thatthe carboxyl group content of the alkali untreated sample was 0.0013(ratio of carboxyl-group carbon with respect to total carbon), whilethat of the alkali-treated sample was 0.0015, showing an increase in thesurface carboxyl group content.

In addition, TOF-SIMS (time-of-flight secondary ion mass spectrometry)analysis on the amount and distribution of fluorine on the surface ofthe two samples of which the surface carboxyl groups were labeled withtrifluoroethanol gave the following results. When two samples werecompared, sodium hydroxide-treated sample showed stronger peaks of ¹⁹F⁻and ⁶⁹CF³⁻ than untreated sample, indicating that the sodiumhydroxide-treated sample contained more carboxyl groups than theuntreated sample. Specifically, an ionic count of the peak having a massnumber 19 corresponding to F⁻ of the untreated sample was 8,000, whilean ionic count of the sodium hydroxide-treated sample was 25,000. Inaddition, the ionic count of the peak having a mass number 69corresponding to CF³⁻ of the untreated sample was 1,200, but that of thesodium hydroxide-treated sample was 7,000. Further, two-dimensional TOFSIMS analysis on the fluorine distribution on the surface of the samplesrevealed that the sodium hydroxide-treated sample had a localized ¹⁹F⁻ion image (presence of circular areas of 30 to 40 μm and streaks of 30to 40 μm in width lower in ionic strength). The results indicate thatthe alkali-treated sample has a distribution in which the surfacecarboxyl groups localized. On the other hand, the untreated sample didnot show the distribution in which the ¹⁹F⁻ ion image is particularlylocalized. Alternatively, both two samples showed no particularlylocalized distribution in the ³¹CH₃O⁻ ion image presumably correspondingto a methoxy group.

Example 1 Preparation of DNA-Immobilized Support

A transparent polymethyl methacrylate (PMMA) plate (Comoglass extrudedplate, manufactured by Kuraray Co., Ltd.; thickness: 1 mm, averagemolecular weight: 150,000, i.e., number-averaged polymerization degree:1,500) was immersed in an aqueous 10N sodium hydroxide solution at 65°C. for 12 hours. Then, the plate was washed thoroughly with purifiedwater, an aqueous 0.1N HCl solution, and purified water in that order.In this manner, carboxyl groups are formed on the plate surface byhydrolysis of the side chains of PMMA. The intensity of theself-fluorescence of the plate (non-alkali-treated) was 650, asdetermined under the conditions of an excitation wavelength of 532 nm, aset photomultiplier gain of 700, and a laser power of 33% by usingGenePix 4000B manufactured by Axon Instruments.

(Immobilization of Probe DNA)

DNA's having sequence No. 1 (70 bases, 5′-terminal aminated), sequenceNo. 2 (60 bases, 5′-terminal aminated), sequence No. 3 (40 bases,5′-terminal aminated), and sequence No. 4 (20 bases, 5′-terminalaminated) were prepared. 5′-Terminals of the DNA's having sequence Nos.1 to 4 were aminated.

These DNA's were dissolved in purified water to a concentration of 0.27nmol/μl, to give respective stock solutions. For spotting on substrate,prepared was a solution of each probe diluted with PBS (a solution of 8g of NaCl, 2.9 g of Na₂HPO₄-12H₂O, 0.2 g of KCl, and 0.2 g of KH₂PO₄dissolved in purified water to a total volume of 1 L and addedhydrochloric acid for pH adjustment, pH: 5.5) to a final concentrationof 0.027 nmol/μl, containing additionally1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) at a finalconcentration of 50 mg/ml, for condensation of the carboxyl groups onthe support surface with the terminal amino group of the probe DNA.Then, each of these mixture solutions was spotted on the substrate in anamount of approximately 200 nl. That is, the four kinds of probes werespotted respectively at one point on the PMMA substrate. Then, thesubstrate was placed in a tightly sealed plastic container, andincubated under the condition of 37° C. and a humidity of 100% forapproximately 20 hours, and then washed with purified water. FIG. 1shows the reaction scheme.

(Preparation of Sample DNA):

A DNA of sequence No. 8 (968 bases) having a base sequence hybridizablewith the DNA-immobilized substrate was used as a sample DNA.

The preparative method is as follows: DNA's of sequence Nos. 5 and 6were prepared. These DNA's were respectively dissolved in purified waterto a concentration of 100 μM. Then, pKF3 plasmid DNA (Takara Bio Inc.Product Number: 3,100, sequence No. 7, 2,264 bases) was made available,and was amplified by using it as template and DNA's of sequences Nos. 5and 6 as primers in a PCR reaction (Polymerase Chain Reaction).

The PCR condition is as follows: ExTaq (2 μl), 10×ExBuffer (40 μl), anddNTp Mix (32 μl) (these reagents were attached to the Product NumberRRO01A manufactured by Takara Bio Inc.), a solution of sequence No. 5 (2μl), a solution of sequence No. 6 (2 μl), and a solution of template(sequence No. 7) (0.2 p. 1) were mixed and diluted with purified waterto a total volume of 400 L. The liquid mixture was divided into fourmicro tubes, and the PCR reaction was performed by using a thermalcycler. The product was purified by ethanol precipitation and dissolvedin 40 μl of purified water. Electrophoretic analysis of part of thesolution after PCR reaction confirmed that the base length of theamplified DNA was approximately 960 bases and the DNA of sequence No. 8(968 bases) was amplified.

Then, a random primer having 9 bases (manufactured by Takara Bio Inc.,Product Number 3802) was dissolved to a concentration of 6 mg/ml, and 2μl of the solution was added to the above-mentioned purified DNAsolution after PCR reaction. The solution was heated at 100° C. andquenched on ice. 5 μl of the buffer attached to Klenow Fragment(manufactured by Takara Bio Inc., Product Number 2140AK) and 2.5 μl of adNTP mixture (containing dATP, dTTP, and dGTP each at a concentration of2.5 mM and dCTP at a concentration of 400 μM) were added thereto.Further, 2 μl of Cy3-dCTP (manufactured by Amersham Pharmacia Biotech,Product Number PA53021) was added. After addition of 10U of KlenowFragment to the solution, the mixture was incubated at 37° C. for 20hours, to give a Cy3-labeled sample DNA. Use of the random primer duringlabeling resulted in fluctuation in the length of the sample DNA. Thelongest sample DNA is the DNA of sequence No. 8 (968 bases).Electrophoretic analysis of part of the sample DNA solution showed themost intensive band in the area approximately corresponding to 960 basesand bands slightly smeared in the area corresponding to shorter baselengths. The product was then purified by ethanol precipitation anddried.

The labeled sample DNA was dissolved in 400 μl of a solution containing1% (w/v) BSA (bovine serum albumin), 5×SSC (5×SSC: 43.8 g of NaCl and22.1 g of trisodium citrate hydrate dissolved in purified water to atotal volume of 1 L; 43.8 g of NaCl and 22.1 g of trisodium citratehydrate dissolved in purified water to a total volume of 5 L wasdesignated as 1×SSC; and the 10×-concentrated solution, 10×SSC,5×-diluted solution, 0.2×SSC.), 0.1% (w/v) SDS (sodium dodecylsulfate)and 0.01% (w/v) salmon sperm DNA (each concentration: finalconcentration), and used as a solution for hybridization.

(Hybridization)

The probe DNA thus obtained was applied on the immobilized substrate forhybridization of the sample DNA. Specifically, 10 μl of the solution forhybridization was applied dropwise onto the support carrying theimmobilized probe nucleic acid prepared above and the support wascovered with a cover glass. In addition, the cover glass was sealed witha paper bond, for preventing vaporization of the hybridization solution.The support was placed in a plastic container and incubated under thecondition of 65° C. and a humidity of 100% for 10 hours. Afterincubation, the cover glass was removed and the support was washed anddried.

(Measurement)

After hybridization, the fluorescence from the substrate surface wasobserved under a fluorescence microscope (Olympus Optics) for evaluationof the presence of the hybridization. Fluorescent emission indicatinghybridization was observed on the entire probe region. The difference inintensity between the fluorescences on the spot and the backgroundexpanded, as the base number increased from 40, to 60 and 70, i.e.,improving the S/N ratio as the base number of probe is increased.

For more quantitative discussion, the support after treatment was thenset on a scanner for DNA chip (GenePix 4000B, manufactured by AxonInstruments), and the fluorescence therefrom was determined under theconditions of a laser output of 33% and a photomultiplier gain of 500.The results are summarized in Table 1. In the Table, the fluorescenceintensity is the average fluorescence intensity on the spot, and thenoise is the average fluorescence intensity in the area surrounding thespot (area where no DNA was spotted).

Comparative Example 1

Tests were conducted while the PMMA substrate in Example 1 was replacedwith a glass substrate.

A slide glass was immersed in an aqueous 10N NaOH solution for 1 hourand washed thoroughly with purified water. Then, APS(3-aminopropyltriethoxysilane; manufactured by Shin-Etsu Chemical Co.,Ltd.) was dissolved in purified water at a ratio of 2% (w/v), and theabove-mentioned slide glass was immersed therein for 1 hour, thenremoved from the solution, and dried at 110° C. for 10 minutes. In thisway, amino groups are introduced on the glass surface.

Then, 5.5 g of succinic anhydride was dissolved in 335 ml of1-methyl-2-pyrrolidone. 50 ml of 1M sodium borate solution (containing3.09 g of boric acid and sodium hydroxide for pH adjustment in purifiedwater to a total volume of 50 ml, pH: 8.0) was added to the succinicacid solution. The glass plate above was immersed in the liquid mixturefor 20 minutes. After immersion, the glass plate was washed withpurified water and dried. In this manner, amino groups on the glassplate surface and succinic anhydride were allowed to react with eachother, introducing carboxyl groups on the glass surface. The resultingglass plate was used as the substrate for DNA immobilization. Further,DNAs having base sequences of 1 to 4 were immobilized respectively onthe glass plate in a similar manner to Example 1. FIG. 7 shows thereaction scheme (in FIG. 7, 2 represents a selective binding substance(DNA), and 5, a glass plate). The glass plate was then hybridized in asimilar manner to Example 1. The plate was examined in a similar mannerto Example 1 under a fluorescence microscope.

Upon observation under the fluorescence microscope, light emission wasobservable also on the glass plate only when the probe region is 40bases or more. However, the fluorescence intensity from the three glassplates in Comparative Examples was shown to be distinctively lower thanthat when the substrate is PMMA. For further quantitative discussion,the light intensity from these substrates was determined by using ascanner. The results thereof and from Example 1 are summarized inTable 1. The results in Table 1 indicate that the fluorescence is lower,the noise larger, and the S/N ratio inferior on the glass plate.

Separately, a DNA was immobilized and hybridized in the same scheme asabove by using another commercially available slide glass carrying aminogroups. The slide glasses used were a coated slide glass carryinghigh-density amino groups for DNA microarray (manufactured by MatsunamiGlass Ind., Ltd., Product Number: SD00011) and a MAS-coated slide glass(manufactured by Matsunami Glass Ind., Ltd., Product Number: S081110).Analysis in a similar manner to above showed that the S/N ratio wasinferior even when these slide glasses were used compared to when a PMMAplate is used as the substrate. The results are summarized in Table

TABLE 1 Base length of probe 70 Bases 60 Bases 40 Bases 20 Bases Kind ofFluorescence Fluorescence Fluorescence Fluorescence substrate intensityNoise intensity Noise intensity Noise intensity Noise Example 1 PMMA23000 150 18400 155 12400 150 2500 145 Comparative APS glass 3000 16001800 1540 1200 1620 1680 1330 Example 1 MAS glass 7500 2000 6700 21002000 1890 1530 1680 Glass carrying 8500 2300 7200 2000 3500 1800 18501500 high-density amino groups

Comparative Example 2 Preparation of Slide Glass

Amino groups were introduced on the surface of a slide glass by using3-aminopropyltriethoxysilane in a similar manner to ComparativeExample 1. Then, carboxyl groups were introduced on the surface of theslide glass in a similar manner to Comparative Example 1 by usingsuccinic anhydride, and then the slide glass was washed withacetonitrile and dried under reduced pressure for 1 hour. After drying,the terminal carboxylated glass plate was immersed in a solution of EDC(955 mg) and N-hydroxysuccinimide (575 mg) in acetonitrile (50 ml) for 2hours, washed with acetonitrile, and dried for 1 hour under reducedpressure, to give a glass plate having N-hydroxysuccinimide groups boundto the surface via ester bonds.

(Immobilization of DNA)

By using a 5′-terminal aminated DNA similar to that in Example 1, 200 nlof an aqueous DNA dispersion in 0.1M carbonate buffer solution (pH: 9.3,DNA concentration: 0.027 nmol/μl) was spotted on the glass plate thusobtained. Immediately then, the glass plate after immobilization wasleft at 25° C. and a humidity of 90%, and the glass plate was washedtwice with a mixture solution of 0.1% (w/v) SDS and 2×SSC and once withan aqueous 0.2×SSC solution successively. Then, the glass plate afterthe washing was immersed in an aqueous 0.1M glycine solution (pH: 10)for 1 hour and 30 minutes, washed with distilled water, and dried atroom temperature, to give a glass plate carrying the immobilized DNAfragment.

(Detection)

The glass plate was hybridized by using the same sample DNA in a similarmanner to the hybridization test in Example 1. Results are summarized inTable 2. As apparent from the results, the substrate in Example 1 has anunsatisfactory S/N ratio, compared to the PMMA substrate.

TABLE 2 Base length of probe 70 Bases 60 Bases 40 Bases 20 BasesFluorescence Fluorescence Fluorescence Fluorescence intensity Noiseintensity Noise intensity Noise intensity Noise Comparative 4300 20003000 1500 1800 1220 1500 1200 Example 2

Example 2 Preparation of DNA-Immobilized Support

Carbon black was mixed with a PMMA having an average molecular weight of150,000 at a ratio of 3 wt %, and the mixture was processed into a blacksubstrate having a thickness of 1 mm by casting method. The black PMMAsubstrate was immersed in an aqueous 10N sodium hydroxide solution at65° C. for 12 hours. The substrate was washed with purified water, anaqueous 0.1N HCl solution, and purified water in that order. Theintensity of the self-fluorescence of the plate (without alkalitreatment) was 250, as determined under the conditions of an excitationwavelength of 532 nm, a set photomultiplier gain of 700, and a laserpower of 33% by using GenePix 4000B manufactured by Axon Instruments. Ablackened DNA-immobilized support was prepared in a similar procedure byusing four kinds of probe DNA's similar to those used in Example 1.

Separately, a similar substrate was prepared and the spectroscopicreflectance and transmissibility of the black substrate were determined,and as a result, the substrate had a spectroscopic reflectance of 5% orless at a wavelength in the entire visible light range (wavelength: 400to 800 nm) and a transmissibility of 0.5% or less at a wavelength in thesame range. The substrate had a uniformly flat spectrum without aparticular spectral pattern (e.g., peaks) both in spectroscopicreflectance and transmissibility in the visible light range. Thespectroscopic reflectance is a spectroscopic reflectance includingregular reflectance from the support, as determined by using a deviceequipped with an optical illuminator-detector system (CM-2002,manufactured by Minolta Camera) compatible with the condition C of JIS Z8722.

(Preparation and Hybridization of Sample DNA)

A sample DNA was prepared and hybridized in a similar manner to Example1.

(Measurement)

Fluorescence was measured with a scanner under the same condition asthat in Example 1. The results of scanner observation are summarized inTable 3. In a similar manner to Example 1 the fluorescence intensityincreased as the base number increased. The background fluorescenceintensity also declined, compared to the result in Example 1. Theresults confirmed that blackening of the substrate lead to decrease innoise and increase in S/N ratio.

TABLE 3 Base length of probe 70 Bases 60 Bases 40 Bases 20 Bases Kind ofFluorescence Fluorescence Fluorescence Fluorescence Substrate intensityNoise intensity Noise intensity Noise intensity Noise Example 2 BlackPMMA 25000 50 21000 45 11800 66 1500 52

Example 3

A slide glass carrying 3-aminopropyltriethoxysilane introduced on thesurface was prepared in a similar manner to Comparative Example 1. PMMAdissolved in chloroform was spin-coated thereon, and the slide glass wasleft at 100° C. for 15 minutes and additionally at 115° C. for 1 hour,to give a support consisting of a support layer (glass) and a layercarrying an immobilized selective binding substance (PMMA). Thethickness of the spin-coated PMMA was approximately 20 μm.

Then, the support was immersed in 10N NaOH for 10 hours, formingcarboxyl groups on the PMMA surface. A probe DNA was immobilized, asample DNA was prepared and hybridized, and the fluorescence intensitythereof was determined in a similar manner to Example 1. Results similarto those in Example 1 were obtained. Although the substrate was slightlybent in Example 1, there was no warp observable in the support of thisExample.

In addition, when the PMMA used in Example 2 containing dispersed carbonblack was spin-coated similarly, the fluorescence intensity and noisesimilar to those in Example 2 were obtained, and there was no warpobservable in the support also in this case.

Example 4

A test was conducted in a similar manner to Example 1, except that 10Nsulfuric acid was used instead of the aqueous 10N NaOH solution duringintroduction of carboxyl groups on the PMMA surface. Consequently,results similar to those in Example 1 were obtained.

Example 5

A copolymer of styrene and MMA (methyl methacrylate) was prepared. Thecopolymer prepared had a composition of 10 mol % MMA and 90 mol %styrene. Specifically, the copolymer was prepared by dissolving MMA andstyrene at a ratio of 1:9 (molar ratio) in dehydrated toluene, addingAIBN (azobisisobutylnitrile) at a ratio of 1/1000 with respect to thetotal mole number of MMA and styrene, and leaving the mixture undernitrogen atmosphere at 60° C. for 1 hour, at 65° C. for 3 hours, andadditionally at 90° C. for 20 hours. The copolymer thus obtained waspurified by ethanol precipitation and filtration.

The composition of the purified polymer was examined by NMR (nuclearmagnetic resonance). The molecular weight of the polymer was determinedby GPC, and the number-averaged polymerization degree calculatedtherefrom was 1,100.

Then, the purified polymer was processed into a plate having a thicknessof 1 mm by casting method. The tests similar to those in Example 1 wereperformed, except that the DNA of sequence No. 2 was used forimmobilization. The fluorescence was determined with a scanner under theconditions similar to Example 1. The result revealed that thefluorescence intensity was 5,200, the noise, 150, and the MMA content,10%, and that the S/N ratio was improved, compared to those inComparative Examples 1 and 2. The self-fluorescence intensity of anon-alkali-treated flat plate was 750, as determined by using GenePix4000B manufactured by Axon Instruments under the conditions of anexcitation wavelength of 532 nm, a set photomultiplier gain of 700, anda laser power of 33%.

Comparative Example 3

A homopolymer of polystyrene was prepared and processed into a platehaving a thickness of approximately 1 mm by casting method. In the testsby using the plate similar to those in Example 1, there was nofluorescence indicating hybridization between the probe DNA and thesample DNA observed at all.

Example 6 Preparation of DNA-Immobilized Support

A mold for injection molding was prepared according to a known LIGA(Lithographie Galvanoformung Abformung) process, and a PMMA substratehaving the shape described below was prepared by injection molding. ThePMMA used in this Example had an average molecular weight of 150,000 andcontained carbon black (#3050B, manufactured by Mitsubishi ChemicalCorp.) at a ratio of 1 wt %, and the substrate was black in appearance.When the spectroscopic reflectance and transmissibility of the blacksubstrate were determined, the spectroscopic reflectance was 5% or lessat a wavelength in the visible light range (wavelength: 400 to 800 nm)and the transmissibility was 0.5% or less at a wavelength in the samerange. The spectra of both the spectroscopic reflectance andtransmissibility were uniformly flat, without particular spectralpatterns (e.g., peaks). The spectroscopic reflectance is a spectroscopicreflectance including regular reflection from the support, as determinedby using a device equipped with an optical illuminator-detector system(manufactured by Minolta Camera, CM-2002) compatible with the conditionC of JIS Z 8722.

The shape of the substrate was 76 mm in length, 26 mm in width, and 1 mmin thickness, and the surface was flat except in the central are of thesubstrate. A concave part of 10 mm in diameter and 0.2 mm in depth 0.2mm is formed on the center of the substrate, and 64 (8×8) projectionshaving a top face diameter of 0.2 mm and a height of 0.2 mm were formedin the concave part. The projections, which have a basal diameter(diameter of the base region of the projection) of 0.23 mm, are tapered,for convenience in releasing the substrate after injection molding. Thedifference between the height of projection top face (average of theheights of 64 projections) in the concavo-convex part and the height ofthe flat area was 3 μm or less, when determined. In addition, thevariation in height of the 64 projection top faces (difference in heightbetween the highest and the lowest projection top faces), and thedifference between the height of projection top face in the irregularlysurfaced area and the height of the flat area, when determined, wereboth 3 μm or less. Further, the pitch of the projections in theirregularly surfaced area (distance between a projection center toanother projection center next to it) was 0.6 mm.

The PMMA substrate was immersed in an aqueous 10N sodium hydroxidesolution at 65° C. for 12 hours. The substrate was washed with purifiedwater, an aqueous 0.1N HCl solution, and purified water in that order,to give a substrate having carboxyl groups formed on the surface. Theself fluorescence intensity of the plate, i.e., the PMMA flat platecontaining carbon black used in this Example, was 250, as determinedunder the conditions of an excitation wavelength of 532 nm, a setphotomultiplier gain of 700, and a laser power of 33% by using GenePix4000B manufactured by Axon Instruments.

(Immobilization of Probe DNA)

The DNA of sequence No. 2 (60 bases, 5′-terminal aminated) was prepared.The DNA was aminated at the 5′-terminal. The DNA was dissolved inpurified water at a concentration of 0.27 nmol/μl, which was used as astock solution. For spotting the substrate, the stock solution wasdiluted with PBS (pH: 5.5) to a final probe concentration of 0.027nmol/μl, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) wasadded thereto to a final concentration of 50 mg/ml for facilitatingcondensation between the carboxyl groups on the support surface and theterminal amino group of the probe DNA. The mixture solution, i.e., probeDNA, was the spotted at four points on the projections of the PMMAsubstrate with a glass capillary under a microscope. Then, the substratewas placed in a tightly sealed plastic container and incubated under thecondition of 37° C. and a humidity of 100% for approximately 20 hours,and then, washed with purified water.

(Preparation of Sample DNA)

The sample DNA was prepared in a similar manner to Example 1.

(Hybridization)

To 10 μL of the DNA solution described above (preparation of sampleDNA), added was 30 μl of a solution containing 1% (w/v) BSA, 5×SSC, 0.1%(w/v) SDS, and 0.01% (w/v) salmon sperm DNA, to make the total solution40 μl (that is, the sample concentration was 1/4 compared to that inExamples 1 or 2, but the total sample quantity is the same as that inExample 1.). The solution was applied dropwise onto the irregularlysurfaced area of the DNA-immobilized support described above, and thesubstrate was covered carefully with a cover glass. Then, the areasurrounding the cover glass was sealed with a paper bond, for preventionof vaporization of the hybridization solution. Namely, the molecularweight of the sample DNA was made the same as those in Example 1 andComparative Example 1. The substrate was placed in a plastic containerand incubated under the condition of a humidity of 100% and atemperature of 65° C. for 10 hours. After incubation, the cover glasswas removed, and the substrate was washed and dried.

(Measurement)

The fluorescence therefrom was analyzed in a similar manner to Example 1with a scanner. The results are summarized in Table 4.

TABLE 4 Spot 1 Spot 2 Spot 3 Spot 4 Kind of Fluorescence FluorescenceFluorescence Fluorescence Substrate intensity Noise intensity Noiseintensity Noise intensity Noise Example 6 Black concavo- 21500 27 2050026 20200 28 20800 22 convex surfaced PMMA

As apparent from the results, the fluorescence intensity was almost thesame as that in Example 2, but the noise was further reduced from thatin Example 2.

Example 7

Subsequently, a test was performed by using a substrate havingprojections irregular in height. The projections on the injection-moldedPMMA substrate used in Example 6 were polished with a polishing paper,to make variation in the height of the projection top faces.Specifically, a support (support A) having four projections lower by 30μm than other projections (standard projection) and a support (supportB) having four projections lower by 50 μm than other projections wereprepared. The difference in height between the face of the projectionsother than the lower projections (standard projection) and the face ofthe flat area was 3 μm or less. A probe DNA for spotting was prepared ina similar manner to Example 6. Then, the probe DNA solution was spottedon the faces of four standard projection and four lower projections in asimilar manner to Example 6. Further, a hybridization DNA was preparedand hybridized in a similar manner to Example 6, and the hybridizedsupport was analyzed in a similar manner to Example 6. Averages of thefluorescence intensities from the faces of standard projections and ofthe noise from the area surrounding them, and the average fluorescenceintensity from the faces of the lower projections and the average noisefrom the surrounding area are summarized in Table 5.

TABLE 5 Result of standard projections Result of lower projectionsFluorescence Fluorescence Kind of Support Intensity Noise IntensityNoise Example 7 Support A 21000 30 18500 26 Support B 20800 25 16800 29

The results show that a significantly larger S/N ratio can be obtainedcompared to that in Comparative Examples even on a substrate where thereis some fluctuation in the height of projections (50 μm or less).

Example 8

In addition, a support having a difference in height between theprojection top face and the flat area was also examined. The projectionson the injection-molded PMMA substrate used in Example 6 were polishedwith a polishing paper, to make two supports respectively havingdifferences in height by 30 μm (support C) and 50 μm (support D) betweenthe faces of the flat area and the projection top face. Namely, thesupport C has projections higher by 30 μm than the flat area. A probeDNA for spotting was prepared and spotted onto the face of theprojections; a DNA for hybridization was prepared and hybridized in asimilar manner to Example 6; and the support was analyzed in a similarmanner to Example 6. The number of the projections in the substrate onwhich the DNA solution was spotted was four. Averages of thefluorescence intensities of the DNA-bound spots (four spots) and thenoises in the areas surrounding them (4 areas) were determined. Theresults are summarized in Table 6.

TABLE 6 Base material C Base material D Fluorescence intensity NoiseFluorescence intensity Noise Example 8 19100 32 16500 30

The results show that a significantly larger S/N ratio can be obtainedcompared to Comparative Examples even where there is some difference inheight between the flat area top face and the projection top face (50 μmor less).

Example 9

Flat plates of polymethyl methacrylate, polyethyl methacrylate, andpolyphenyl methacrylate were prepared by casting and immersed in anaqueous 10N sodium hydroxide solution at 50° C. for 10 hours. The flatplates were washed with purified water, an aqueous 0.1N HCl solution,and purified water in that order. In this way, carboxyl groups wereformed on the surface of the plates by hydrolysis of the polymerside-chains.

A probe DNA was immobilized (the immobilized probe DNA had only 60bases); a sample DNA was prepared and hybridized; and the resultingplate was analyzed in a similar manner to Example 1. The results aresummarized in Table 7.

TABLE 7 Kind of support Fluorescence intensity Noise Example 9Polymethyl 13100 175 methacrylate Polyethyl 12000 190 methacrylatePolyphenyl 15000 350 methacrylate

These results show that the flat plates show significantly large S/Nratios, compared to Comparative Examples.

Example 10 Preparation of DNA-Immobilized Support

A substrate similar to that in Example 6 was prepared. Then, a Ni—Crfilm (Ni/Cr composition is Ni₈Cr₂) having a thickness of 50 nm wasformed on the substrate by sputtering. Separately, a solution of 1 g ofpulverized granules of the substrate (without a Ni—Cr film) dissolved in10 mL of chloroform was prepared. Then, an insulation layer of blackPMMA film was formed on the Ni—Cr film by coating the solution thereonby spin coating. After removal of the insulation layer and the Ni—Crfilm on the projection top face with a polisher, the substrate wasimmersed in 10N NaOH solution at 65° C. and then washed with purifiedwater, an aqueous 0.1N HCl solution, and purified water in that order. Aprobe DNA was then immobilized, and a sample DNA was prepared in asimilar manner to Example 6.

(Hybridization)

Chromium and gold films were deposited on a cover glass respectively toa thickness of 5 nm and 100 nm. A gold wire was connected thereto bysoldering. Separately, 50 μl of the solution for hybridization was addedonto the irregularly surfaced area of support where the nucleic acidpreviously prepared is immobilized, and the cover glass above was laidon the irregularly surfaced area with its golf face facing the support.At the time, a spacer of 0.2 mm in thickness was placed between thesupport and the gold cover glass for prevention of short circuiting. Thearea surrounding the cover glass was sealed with a paper bond forprevention of vaporization of the hybridization solution.

Then, the Ni—Cr film on support and the anode of power source wereconnected to each other and the gold (gold wire) of cover glass to thecathode of the power source by using a gold wire and a commerciallyavailable silver paste for electrical connection. The support was placedin an oven and incubated therein at 65° C. for 15 minutes. Afterapplication of a voltage of 1 V from the power source for 5 minutes, thesupport was removed from the oven and, after removal of the cover glass,washed and dried.

Consequently, results similar to those in Example 6 were obtained. Inthis manner, even if the hybridization period is shorter, it is possibleto shorten the hybridization period by forming an electrode to the sideface of the projection and applying an electric field.

Comparative Example 4

A plate of 1 mm in thickness having polyacrylonitrile as the maincomponent (Zexlon, manufactured by Mitsui. Chemicals, Inc.) was cut intopieces of 75 mm×25 mm in size. These pieces were immersed and left in10N NaOH at 70° C. for 12 hours. After washing, a probe DNA wasimmobilized (probe DNA length: 60 bases), and the immobilized plate washybridized with a sample DNA in a similar manner to Example 1. The platewas evaluated in a similar manner to Example 1, but it was not possibleto determine whether the sample and the probe were hybridized because ofgreater autofluorescence. It is because the plate is yellowish as it isand extremely higher in autofluorescence. When measured under theconditions of an excitation wavelength of 532 nm, a set photomultipliergain of 700, and laser power of 33% by using GenePix 4000B manufacturedby Axon Instruments, the self-fluorescence intensity of the flat platebefore alkali treatment was extremely large at 30,000.

Comparative Example 5

99 Parts by weight of methyl methacrylate (MMA) and 1 part by weight ofmethacrylic acid were copolymerized. After purification, the copolymerwas dissolved in a solvent, and a film of this polymer was formed on aPMMA plate by dipping. A probe DNA (60 base length) was immobilized onthe film; a sample DNA was hybridized in a similar manner to Example 1,except that the alkali immersion was eliminated; and the resultingsupport was analyzed in a similar manner to Example 1. As a result, thesignal intensity was 6000, and the noise intensity was 300.

Example 11

99 Parts by weight of methyl methacrylate (MMA) and 1 part by weight ofmethacrylic acid were copolymerized. After purification, the copolymerwas dissolved in a solvent, and a film of this polymer was formed on aPMMA plate by dipping. A probe DNA (60 base length) was immobilized onthe film; a sample DNA was hybridized in a similar manner to Example 1,except that the support was immersed in the alkali solution at 50° C.for 10 hours; and the resulting support was analyzed in a similar mannerto Example 1. As a result, the signal intensity was 15,000, and thenoise intensity was 150. The reason for that the results in this Examplewere superior to those in Comparative Example 5 seems to be the presenceof an alkali treatment that increases the amount of carboxyl groups onthe support surface. A plate of 1 mm in thickness of a copolymerprepared from 99 parts by weight of methyl methacrylate (MMA) and 1 partby weight of methacrylic acid was prepared by casting; and when measuredunder the conditions of an excitation wavelength of 532 nm, a setphotomultiplier gain of 700, and a laser power of 33% by using GenePix4000B manufactured by Axon Instruments, the self fluorescence intensityof the non-alkali-treated flat plate was 850.

Comparative Example 6

The same test was repeated, except that the alkali treatment (immersionin a sodium hydroxide solution) in Example 1 was eliminated. As aresult, there was no observed fluorescence indicating hybridization. Itis seemingly because absence of alkali or acid surface treatment resultsin formation of carboxyl groups in an insufficient amount, consequentlyleading to decrease in the amount of a probe DNA immobilized.

INDUSTRIAL APPLICABILITY

We provide a support carrying an immobilized selective binding substancethat is smaller in adsorption of nonspecific samples, favorable inhybridization efficiency, and consequently favorable in S/N ratio.

1. A support carrying an immobilized selective binding substance,wherein the support having a surface with a polymer comprising alow-autofluorescence resin, the support surface of the polymer beingtreated with an alkali or acid, forming carboxy groups and a selectivebinding substance is immobilized on the support surface through thecarboxyl groups.
 2. The support according to claim 1, in which thesupport has a concavo-convex portion made up of multiple projections andcarrying the selective binding substance immobilized on a surface of topfaces of the multiple projections.
 3. The support according to claim 1,in which the low-auto fluorescence resin has a fluorescence intensity of1,000 or less.
 4. The support according to claim 1, in which thelow-autofluorescence resin is a homopolymer selected from the group ofpolyalkyl methacrylate, polyvinyl acetate, polycyclonexyl methacrylateand polyphenyl methacrylate, or a copolymer comprising polymercomponents thereof.
 5. The support according to claim 1, in which thelow-autofluorescence resin is polyalkyl methacrylate or a copolymercomprising alkyl methacrylate in amount of 10% or more with respect tothe total monomer units.
 6. The support according to claim 1, in whichthe low-autofluorescence resin is polymethyl methacrylate.
 7. Thesupport according to claim 1, in which the immobilized selective bindingsubstance is immobilized on the top faces of the multiple projectionsthrough the carboxyl groups with a condensation agent.
 8. The supportaccording to claim 1, in which the selective binding substance isimmobilized by a covalent bond between the amino or hydroxyl group ofthe selective binding substance and a carboxyl group on the supportsurface.
 9. The support according to claim 1, in which the selectivebinding substance is a nucleic acid.
 10. The support according to claim1, in which the support surface is black in color.
 11. The supportaccording to claim 1, in which the low-auto fluorescence resin containscarbon black.
 12. The support according to claim 1, in which the supporthas at least a support layer and a layer carrying an immobilizedselective binding substance, a surface of the layer carrying animmobilized selective binding substance has a polymer comprising thelow-autofluorescence resin, and the selective binding substance is boundto the surface of the selective binding substance-immobilized layer. 13.The support according to claim 12, in which the support layer is glassor a metal.
 14. The support according to claim 1, in which the top facesare substantially flat and the top faces are almost the same height. 15.The support according to claim 1, in which the support has a flat areaformed.
 16. The support according to claim 1, in which the differencebetween the heights of the highest and the lowest projections amongmultiple projections carrying an immobilized selective-binding substrateis 50 μm or less.
 17. The support according to claim 15, in which thedifference in height between the face of the projections in theconcavo-convex part and the face of the flat area is 50 μm or less.